Neuronal haemoglobin induces loss of dopaminergic neurons in mouse Substantia nigra, cognitive deficits and cleavage of endogenous α-synuclein.

Parkinson's disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. α-synuclein (α-syn) species truncated at the carboxy-terminal (C-terminal) accumulate in pathological inclusions and promote α-syn aggregation and toxicity. Haemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells' vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. In this study, α and ß-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. Long-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, mild motor impairments, and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous α-syn C-terminal truncated species. Similar α-syn fragments were found in vitro in DA iMN9D cells over-expressing α and ß- globins when treated with pre-formed α-syn fibrils. Our study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells' loss in vivo and the formation of C-terminal α-syn fragments.

SINEUP non-coding RNAs rescue defective frataxin expression and activity in a cellular model of Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases.

Castanea sativa Mill. Bark Extract Protects U-373 MG Cells and Rat Brain Slices Against Ischemia and Reperfusion Injury.

Ischemic brain injury is one of the most important causes of death worldwide. The use of one-drug-multi-target agents based on natural compounds is a promising therapeutic option for cerebral ischemia due to their pleiotropic properties. This study assessed the neuroprotective properties of Castanea sativa Mill. bark extract (ENC) in human astrocytoma U-373 MG cells subjected to oxygen-glucose deprivation and reperfusion and rat cortical slices subjected to ischemia-like conditions or treated with glutamate or hydrogen peroxide. Neuroprotective effects were determined by assessing cells or slices viability (MTT assay), ROS formation (DCFH-DA assay), apoptosis (sub G0/G1 peak), nuclear fragmentation and chromatin condensation (DAPI staining) as well as changes in lysosomes and mitochondria morphology (Acridine Orange and Rhodamine123 staining, respectively). ENC treatment before injury on U-373 MG cells (5-50 µg/ml) and cortical slices (50-100 µg/ml) provided neuroprotection, while lower or higher concentrations (100 µg/ml U-373 MG cells, 200 µg/ml brain slices) were ineffective. ENC addition during reperfusion or after the injury was not found to be effective. The results suggest that ENC might hold potential as preventive neuroprotective agent, and indicate the importance of further studies exploring its mechanism of action. J. Cell. Biochem. 118: 839-850, 2017. © 2016 Wiley Periodicals, Inc.

Neuroprotective Effects of Castanea sativa Mill. Bark Extract in Human Neuroblastoma Cells Subjected to Oxidative Stress.

One of the major features of neurodegenerative disease is the selective vulnerability of different neuronal populations that are affected in a progressive and often stereotyped manner. Despite the susceptible neuronal population varies between diseases, oxidative stress is implicated as the major pathogenic process in all of them. Natural Extract of Castanea sativa Mill. bark (ENC), recently characterized in its phenolic composition, acts as antioxidant and cardioprotective agent. Its neuroprotettive properties, however, have never been investigated. The aim of this study was to assess neuroprotection of ENC in in vitro models of oxidative-stress-mediate injury. Human neuroblastoma SH-SY5Y cells treated with glutamate (50 mM for 24 h) or hydrogen peroxide (25 µM for 1 h followed by 24 with medium) were used. The results showed that the addition of ENC (1-50 µg/ml) to cell medium before the neuronal damage provided neuroprotection in both experimental models used, while its addition after the injury was ineffective. In conclusion, the present results suggest that ENC could be a valuable support as dietary supplement, combining beneficial preventive neuroprotettive effects with a high antioxidant activity.